During the past decade, it has become evident that in vitro enzyme data obtained in solution frequently fail to provide an accurate profile of in vivo enzyme kinetic parameters. This may be due to oligomer formation. Efforts to separate the effect of oligomer formation on P450 kinetics from factors that might influence kinetic parameters have been incomplete and are necessary to quantify the contribution that oligomer formation has on P450-mediated metabolism kinetics, to improve estimations of in vivo drug clearance. Our long-term goal is to accurately predict in vivo drug pharmacokinetics from in vitro data to improved assessments of drug safety and efficacy. Our overall objective here is to determine the important factors influencing in vitro P450-mediated metabolism kinetics through studies of homo- and hetero-oligomer formation using our recently developed immobilized enzyme methods. Our central hypothesis is that CYP2C9-mediated metabolism kinetics measured in vitro are critically dependent on the state of oligomerization, and to accurately predict in vivo pharmacokinetics using in vitro data requires inclusion of this variable. Our rationale for this project is that successful completion would provide a solid scientific foundation to more accurately assess in vivo kinetics of metabolism based upon in vitro data. To achieve our overall objectives, we propose to quantitate the modulatory effects of CYP2C9 homo and hetero-oligomer formation on enzyme kinetics. Successful achievement of this goal will well position us to determine the key factors that explain altered enzyme kinetics of oligomers versus monomers. The proposed research is innovative as a novel immobilized P450 methodology will be employed to unequivocally evaluate the effect of oligomerization on the kinetics of metabolism. Our expected outcomes are 1) determination of selected physical-chemical factors that affect CYP2C9 kinetics using nanoscale methods for protein manipulation and 2) to have quantitated the extent to which oligomer formation modulates CYP2C9-mediated metabolism kinetics. These outcomes are significant as they will help to further delineate the mechanistic details of P450 metabolism, an enzyme system responsible for metabolism of the majority of drugs, and should lead to improved pharmacokinetic data, necessary for determination of correct dosages and thus ultimately improve patient care.